To understand the interleukin 1 converting enzyme (ICE), it is helpful to first examine the role of interleukin-1 (IL-1), its enzymatic substrate. IL-1 facilitates host natural immunity, predominantly those aspects related to the initiation of inflammatory reactions that protect the body against bacterial infection. (Ayala et al. (1994) J. Immunol. 53: 2592-2599). At low concentrations in the bloodstream, IL-1 mediates local inflammation by inducing the synthesis of other cytokines, such as IL-6 and IL-8, and the synthesis of proteins that mediate leukocyte adhesion, and prostaglandin production. (Abbas et al. (1994) Cellular and Molecular Immunology, W.B. Saunders Company). Whereas at intermediate concentrations, IL-1 enters the bloodstream and may induce fever, the synthesis of acute plasma proteins by the liver, and metabolic wasting (cachexia) (Abbas, supra). At even higher concentrations, IL-1 has been implicated in tissue destruction observed in numerous inflammation-related diseases, including rheumatoid arthritis, septic shock, inflammatory bowel disease and insulin-dependent diabetes mellitus. (Li et al. (1995) Cell 80:401-411).
IL-1 activity results from the expression and release of two gene products, IL-1.alpha. and IL-1.beta., predominantly from activated monocytes. (Howard et al. (1991) J. Immunology 147:2964-2969). Both gene products are initially synthesized as inactive precursors of about 31 kDa in monocytes. Pre-IL-1.beta. is cleaved to an active 17 kDa form by the IL-1.beta.-converting enzyme (ICE) before release from activated monocytes. On the other hand, pre-IL-1.alpha. is likely cleaved to an active 17 kDa form by a calpain-like IL-1.alpha.-converting enzyme prior to release. (Carruth et al. (1991) J. Biol. Chem. 266:12162-12167). Additionally, ICE has been implicated in IL-1.alpha.'s release from activated monocytes but the mechanism is not understood (Li, supra).
The IL-1.beta. gene product is the predominant form of IL-1 that is present at high concentrations in the bloodstream during inflammatory diseases, such as rheumatoid arthritis, septic shock, inflammatory bowel disease, and insulin-dependent diabetes mellitus (Li, supra). Since the cleavage of pre-IL-1.beta. by ICE is coupled to IL-1.beta. release and to increased IL-1 activity in the bloodstream, ICE activity may be higher in these pathological conditions.
The importance of regulating ICE activity to modulate the IL-1.beta. concentration to affect the host immune response has recently been confirmed: the crmA gene product of cowpox virus prevents the proteolytic activation of IL-1.beta. and inhibits the host inflammatory response. Cowpox virus containing a deleted crmA gene is unable to suppress the inflammatory response, resulting in a reduction of virus-infected cells and less damage to the host. (Miura et al. (1993) Cell 75: 653-660).
ICE is a novel cysteine protease that is known specifically to cleave inactive IL-1.beta. precursor to its active form. (Ayala, supra). This protease recognizes the sequence Asp-X, where X is preferably a small hydrophobic amino acid residue, and cleaves the bond between Asp and X. However, many Asp-X bonds are not recognized by ICE suggesting that flanking sequences or other criteria such as accessibility are also required for recognition and cleavage. In the case of IL-1.beta., ICE cleaves the precursor to form active IL-1.beta. at two sequence-specific bonds: the bond between residues Asp-27 and Gly-28 and the bond between residues Asp-116 and Ala-117.
ICE itself is synthesized and maintained in cells as an inactive 45 kDa precursor which is processed into the active ICE consisting of 20- and 10-kDa subunits, p20 and p10. (Ayala, supra). Both subunits are derived from the 45 kDa precursor which is cleaved into four different fragments: a 13 kDa precursor domain, the p20, a 2 kDa spacer, and the p10. Since all these polypeptide fragments are flanked by Asp-X residues in the intact 45 kDa precursor it is possible that the ICE precursor is activated autocatalytically. (Ayala, supra).
The three dimensional structure of ICE has been determined from crystallographic studies. (Walker et al. (1994) Cell 78:343-352). First, it is apparent that the active form of ICE is a homodimer of catalytic domains, each of which consists of p20 and p10 subunits. Second, although the active site cysteine residue is located on p20, both p20 and p10 are essential for activity. p20 and p10 structures are intertwined so as to create a unique 6-stranded beta-sheet core flanked on either side by alpha helices. The first 4 beta strands are contributed by p20, while the remaining 2 beta strands are contributed by p10.
The ICE gene from various sources has been sequenced and possesses homology (overall 29% homology) to the product of a gene with a possible role in apoptosis: the Caenorhabditis elegans gene ced-3. (Yuan et al. (1993) Cell 75: 641-652). Additionally, the ICE gene contains a sequence region, spanning residues 166 to 287 of the human ICE gene, which shares a 43% homology with ced-3. It is not known whether ced-3 acts as a cysteine protease but it contains the purported catalytic residues that are located at the ICE active site (Cys.sub.285 and His.sub.237). The amino acid pentapeptide Glu-Ala-Cys-Arg-Gly (QACRG), containing the active site cysteine, is the longest peptide conserved among ICE from mice and humans and CED-3 from three different nematodes. Additionally, ced-3 contains the same four residues whose side chains are implicated in binding the aspartate carboxylate group of the substrate at the catalytic site (Arg-179, Gln-283, Ser 347, and Arg-341) (Yuan, supra).
Inhibition with the ICE-specific inhibitor crnA blocks TNF- and FAS-induced apoptosis. Therefore, ICE or a homolog is believed to be involved in TNF- and FAS-induced apoptosis.
Additionally, ICE possesses a degree of homology to a gene product with a possible role in embryogenesis. The mammalian gene Nedd-2/Ich-1 is expressed during embryonic brain development and is down-regulated in the adult brain. (Yuan, supra). Nedd-2, ced-3, and ICE gene products are about 27% homologous with the carboxy terminal of CED-3 and p10 possessing the highest degree of homology to Nedd-2. The Nedd-2 gene product does not contain the same highly conserved cysteine nor the highly conserved QACRG pentapeptide so Nedd-1 probably is not a cysteine protease.
To confirm ICE's role in inflammation-related diseases by controlling the levels of active IL-1.beta., ICE-deficient knockout mice were created (Li, supra). These genetically-engineered mice were normal physiologically but lacked the ability to process precursor IL-1.beta. to its active form when monocytes were activated with microbial products, such as lipopolysaccharide (LPS). Additionally, the production of IL-1.alpha. was decreased, and the level of other cytokines, tumor necrosis factor (TNF) and IL-6, involved in inflammatory responses to microbial products was somewhat reduced. These mice were resistant to the lethal effects of septic shock when exposed to LPS (Li, supra). Therefore, inhibiting ICE activity to lower the concentration of IL-1.beta. in the bloodstream may be a method of treating inflammation-related diseases. ICE also may help identify patients who are susceptible to these diseases.
Since ICE shares sequence homology to ced-3 and overexpression of ICE appears to induce apoptosis, the ICE-deficient mice studies were important because the mice seemed normal in terms of their development. If ICE played a strong role in apoptosis during development, the ICE-deficient mice should have had gross abnormalities in brain, gut, lymphoid and brain tissues (Li, supra). However, ICE may perform functions other than IL-1.beta. precursor cleavage. ICE mRNA has been detected in a greater variety of tissues than IL-1.beta. mRNA has (Miura, supra).
ICE has attracted interest as a target for novel anti-inflammatory drugs, because the cytokine which it activates, IL-1.beta., is proinflammatory and has been implicated in the pathophysiology of various diseases, including rheumatoid arthritis, septic shock, inflammatory bowel disease and insulin-dependent diabetes mellitus (Dinarello and Wolff (1993) N Engl J Med 328:106-13). The provision of a new ICE gene and polypeptide will further drug research in screening for and designing more effective and more specific inhibitors to this pro-inflammatory substance.
THP-1 Cells
THP-1 is a human leukemic cell line with distinct monocytic characteristics derived from the blood of a 1-year-old boy with acute monocytic leukemia (Tsuchiya S. et al (1980) Int J Cancer 26:171-176). The monocytic nature of THP-1 was established using the following cytological and cytochemical criteria: 1) a-naphthyl butyrate esterase activity which could be inhibited by NaF (sodium fluoride), 2) production of lysozyme, 3) phagocytosis (the engulfing of extracellular materials) of latex particles and sensitized sheep red blood cells, and 4) ability of mitomycin C-treated THP-1 cells to activate T-lymphocytes following concanavalin A treatment. Morphologically, the cytoplasm contained small azurophilic granules, the nucleus was indented and irregularly shaped with deep folds, and the cell membrane had Fc and C3b receptors which probably function in phagocytosis.
Typical monocytes develop from monoblasts through promonocytes in the bone marrow and in their mature form have a half-life of approximately three days. Roughly 75% of the circulating monocyte pool is found along the walls of blood vessels although these cells randomly migrate into tissues and become antigen-presenting or phagocytic. Antigen-presenting monocytes include interdigitating reticular and follicular dendritic cells of the lymph nodes and skin. Phagocytic monocytes are prominent as Kupffer cells of the liver and in the lung alveoli and bone marrow.
Whereas precursor monocytes are rich in azurophilic, peroxidase-containing cytoplasmic granules, macrophages have more numerous cell surface receptors by which they monitor their environment. These include receptors for immunoglobulin, complement, growth factors, lipoproteins, peptides and polysaccharides. Binding of ligands to these receptors triggers macrophage proliferation, chemotaxis, secretion and phagocytosis.
Many human myeloid and myelomonocytic cell lines retain some ability to differentiate into more mature phenotypes in response to various internal stimuli including growth factors, lymphokines, cytokines, vitamin D derivatives, and tumor promoters and external agents such as trauma, smoking, UV irradiation, asbestos exposure, and steroids. THP-1 cells treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13 acetate (TPA) are induced to stop proliferating and differentiate into macrophage-like cells which mimic native monocyte-derived macrophages both morphologically and physiologically.
These monocyte/macrophage-like cells exhibit changes in gene expression such as the coinduction of C-fos and c-jun and the down-regulation of c-myb (Auwerx J. (1991) Experientia 47:22-31), increase in density of the complement C3b receptor, and decrease in both FcR and the adhesion molecule, CD4. In addition, THP-1 cells produce lipoprotein lipase and apolipoprotein E associated with atherosclerotic lesions, secrete several proinflammatory cytokines, including IL-1.beta. and TNF (Cochran FR and Finch-Arietta MB (1989) Agents and Actions 27:271-273), and may elaborate powerful oxidants and tissue destroying proteases, such as the IL-1 converting enzyme.